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Alex's MCD Practical
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MCD Practicals Alexandra Burke-Smith
1
MDL1-1 – Spectrophotometry and electrophoresis of haemoglobin Dr James Pease ([email protected])
1. Explain the principles of spectrophotometry including the Beer-Lambert Law.
2. Give examples of classes of biologically important compounds which absorb in the UV and visible ranges, and
distinguish those ranges on the basis of wavelength.
3. Construct a standard curve and use it to determine the concentration of an unknown substance by
spectrophotometry.
4. Discuss the differences between oxy-, deoxy- and met-haemoglobin and their physiological significance.
5. Explain the principles of electrophoresis and explain why HbA and HbS can be separated by electrophoresis, with
reference to the individual mutations
Electrophoresis: method of analysing molecules on the basis of charge, by measuring their migration in an electric
field
Spectrophotometry: way of analysing molecules on the basis of
their spectral properties
Setting up Electrophoresis
Electrophoresis buffer at pH8.6 so that Hb has an overall
negative charge and will migrate towards the anode
(positive)
The cellulose acetate strip is suspended above the buffer
but electrically connected by “wicks” of wet filter paper
(there are plastic rods keep strip in contact with wicks)
E.m.f = 200v, A= 20mA
Spectrophotometry
Spectrophotometer: measures the proportion of light absorbed compared with a blank, expressed as a logarithmic
number known as ABSORBANCE or optical density. The measurement can be made at any wavelength, but needs to
be adjusted for the wavelength required and then calibrated using a blank solution.
Absorbance is the fraction of incident light absorbed by the solution which is measured. It can also be written as optical density/extinction, and is defined as:
A = log10 (light transmitted through blank solution / light transmitted through test solution)
Colours Transmitted and Absorbed Light
When white light is passed through a coloured solution (e.g. Hb),
certain wavelengths of light are selectively absorbed
The colour of the solution is that of the remaining light, which is
transmitted
The MAXIMUM absorbance of coloured solution thus occurs in the
region of opposite colour:
o Orange solution – max absorption in blue region (and vice
versa)
o Green solution – max absorption in the red region
o Purple solution – max absorption in the yellow region
Colour E.g. of wavelength
Not visible – UV 350
Violet 400
Blue 450
Cyan 500
Green 550
Yellow 600
Orange 650
Red 700
Not visible - IR 750
buffer buffer
cellulose
acetate
strip
filter
paper
“wick”
filter
paper
“wick”
Aerial view of electrophoresis system
MCD Practicals Alexandra Burke-Smith
2
Effect of Oxygen binding state on spectrum of haemoglobin
Most proteins are transparent to visible light, though they
absorb in the ultraviolet range. Haemoglobin is an exception
because there is a haem group associated with the protein
Globin.
This is an unsaturated organic molecule complexed to the
protein which stabilises an Fe2+ ion to form a site which O2 can
bind to reversibly, forming haemoglobin
Binding alters the electron resonance properties of the haem,
changing its absorbance spectrum.
Normal O2 binding does not change the oxidation state of the iron atom. However it can become oxidised to
Fe3+, in which case the molecule is known as methaemoglobin. This is the state which gives dried blood its
brown colour.
Using the spectrophotometer to determine concentration
The Beer-Lambert Law shows that the absorbance of a solution is proportional to the concentration of the
absorbing solute, and to the distance (or path length) travelled by the light through the sample. This can be
expressed mathematically by:
A = E c ℓ
o A = absorbance
o C= concentration of the absorbing substance (typically in mol/litre)
o ℓ = path length (in cm)
o E = constant (the Extinction Coefficient) for the substance being measured at that particular
wavelength.
Since many substances absorb light at some wavelength (often UV), spectrophotometry is a generally useful
way to measure concentration, by exploiting the Beer-Lambert Law.
o If a graph of absorbance against concentration is drawn, unknown concentrations can be
determined
If the substrate or product of an enzyme reaction absorbs light then the method can be used to follow the
course of the reaction.
Note that some substances fail to obey the Beer-Lambert Law (for example, at high concentrations a protein
might form dimers with a different extinction coefficient) and linearity needs to be checked by constructing
a standard curve).
Analysis of Electrophoresis
There will be a difference in motility of HbA, and HbS, due to a difference in charge. This allows them to be
separated by electrophoresis
This difference in charge is due to a point mutation occurring in one amino acid of the β-chain. In this
mutation, the amino acid glutamate in the normal protein (negatively charged) is replaced by valine
(hydrophobic uncharged).
The HbA will migrate further towards the anode due to the increased negative charge.
In this experiment the migration of two otherwise identical proteins depended only on the charge.
Another common technique is gel electrophoresis where proteins migrate through the pores of a gel.
o If the charge differences of proteins can be masked (by including a substance called SDS in the
electrophoresis buffer) the speed of migration depends only on protein size, with the smaller
proteins migrating faster since they pass more easily through the pores.
Spectrum of haemoglobin
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
450 500 550 600 650
Wavelength (nm)
Ab
so
rba
nc
e
oxy-Hb
deoxy-Hb
MCD Practicals Alexandra Burke-Smith
3
o This is a surprisingly accurate method of measuring the molecular mass of a protein in relation to
known standards. The same principle is used to separate nucleic acids on the basis of size.
MDL2-1 – Microscopy of blood cells Dr Peter Clark ([email protected])
1. Demonstrate how to set up a microscope for optimum performance.
2. Recognise the limitations in examining a specimen at different magnifications in terms of area observed and level
of detail.
3. Identify individual cells at the light microscope level at different magnifications.
4. Identify on the basis of a staining technique the major types of white cells and give their relative proportions in a
normal blood sample.
Setting up a light microscope
Step 1: Focus the image
o The main focussing knob (D) may consist of an outer and
inner part for coarse and fine focus, or there may be a
single focussing knob which gives fine focus for the first
quarter turn or so and then coarse focus engages.
Step 2: Focus the condenser
o Close the field iris (E) and adjust the condenser focus knob
(F) until you get a sharp image of the edge of the disc of
light coming through the iris diaphragm. This should be
roughly in the centre of the field of view - if it is not, adjust
it with the centring screws (F).
Step 3: Adjust the field iris
o Open the field iris (E) again until the whole field of view is illuminated.
Step 4: Adjust the condenser iris
o Start with the condenser iris (H) fully open and then close it until it only just begins to darken the
image.
The basis of the method comes from the underlying optical physics and the parameters that theoretically
affect the resolution of a microscope:
o The wavelength of light
o The optical quality of lenses and all other components in the light path
o The refractive index of the medium through which the light passes (which is why the highest
resolution lenses are designed to use oil instead of air between the objective lens and the slide)
o The physical properties of the objective lens (actually a property called the numerical aperture
which is written on the lens next to its magnification, e.g. 10/0.25 means that lens has 10
magnification and a numerical aperture of 0.25. It follows that higher magnification does not
necessarily mean higher resolution, though in fact it is normally the case that higher power lenses
have better numerical apertures)
o The illuminating cone of light provided by the condenser lens (which depends in part on the design
properties of the lens, but to a large degree on the way in which the condenser is focussed and the
iris diaphragms adjusted)
In practice the only adjustment you can make to optimise resolution – short of buying a new microscope – is
to set up the condenser properly.
MCD Practicals Alexandra Burke-Smith
4
Use of stains in microscopy
Very few cellular components absorb light at visible wavelengths so in order to see cells we stain them with
coloured dyes. H&E is the most frequently used combination of stains.
Haematoxylin is a purple-blue component that binds to acidic components of cells. In particular it binds DNA
and shows up the nucleus.
o Many stains (including Leishman’s stain used later in this practical) contain a similar component that
makes the nucleus visible.
Eosin is a pinkish stain that binds protein components, particularly in the cytoplasm.
There are many other stains that can be used to examine cells in thin sections (“slices”) through tissues. H&E
is a good general purpose stain, but others may be chosen for demonstrating particular chemical
constituents of cells, such as carbohydrate.
Modern methods allow one to investigate the location of a specific molecular component.
The alternative to using stains is to exploit interference properties of light to detect the different refractive
index of cells compared with the surrounding fluid.
o This approach (which needs a microscope fitted with special optics) is especially important when
examining living cells.
Examining a blood smear
Obtain a drop of blood from your finger by using a sterile lancet to puncture the fleshy pad alongside the
base of a fingernail.
Immediately place the drop on a clean slide (wipe off dust if necessary using a tissue) and slowly smear it out
in a film using a second slide as a spreader, as
indicated below.
the direction of spreading is so that the blood
drop remains behind the spreader, otherwise
blood cells might become damaged between the
two slides.
Dry the blood smear by gently waving the slide
around in the air, and check the blood film looks
thin and even
Safety considerations
clean your skin with an alcohol swab before puncture
do not share lancets, and dispose of them in the special “sharps” bin provided
protect the wound with a plaster
wear a lab coat, and put on protective gloves to handle any blood other than your own
wash your hands immediately if they accidentally come into contact with blood
never under any circumstance use a mouth pipette
spills of blood must be decontaminated immediately (ask a demonstrator)
at the end of the experiment dispose of glassware and anything which has come into contact with blood in
the sharps bin
MCD Practicals Alexandra Burke-Smith
5
Identification of Blood Cell Types
Non-nucleated cells
(their precursors in the bone marrow had nuclei, but these were lost before they reached the blood stream)
Red blood cell (erythrocyte)
Most abundant blood cell,
involved in O2 transport.
Biconcave shape, dusky pink.
7 m diameter.
Blood platelets
These are important in
blood clotting, and will
probably be found in
small clumps on a
smear. Bluish.
2-4 m diameter.
Nucleated cells
(also known as white blood cells, easily identified by their blue staining nuclei)
Lymphocyte
Small cells with near
spherical nucleus and
little cytoplasm. Involved
in the immune response.
Around 8 m diameter.
Eosinophil
Cytoplasmic granules are
bright pink. Bi-lobed
nucleus. Implicated in
fighting parasitic infections
and allergy. Around 12 m
diameter.
Neutrophil
Multi-lobed (“poly-
morphonuclear”)
nucleus, cytoplasmic
granules. Phagocytic cells
which kill bacteria. 10-12
m diameter.
Basophil
Cytoplasmic granules
staining strongly blue.
Lobed nucleus. Involved in
inflammation and allergic
responses. 10-15 m
diameter.
Monocyte
Indented nucleus and
pale granules in the
cytoplasm. Phagocytic
cells with role in the
immune response and
inflammation. 17-20 m
diameter.
MCD Practicals Alexandra Burke-Smith
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MDL1-2 – Determination of red blood cell parameters Prof. Karim Meeran ([email protected])
1. Work safely with blood.
2. Estimate a red cell count of a blood sample using a microscope and Neubauer haemocytometer slide.
3. Measure haematocrit of a blood sample centrifugally.
4. Measure the haemoglobin concentration of a blood sample using an optical density method.
5. Calculate the MCV, MCH and MCHC using the parameters measured in 2, 3 and 4 above.
6. State the normal ranges for males and females of whole blood red cell count, haematocrit and haemoglobin
concentration and recognise that the other parameters are not gender-dependent
7. State the definition of anaemia and explain why the combined red cell parameters can give an important guide to
the type of anaemia present.
Safety Considerations
Remember that any fresh blood or blood product must be regarded as hazardous material, and any puncture wound
as a possible route of infection. You must therefore take suitable precautions:
clean your skin with an alcohol swab before puncture
do not share needles, and dispose of them in the special “sharps” bin provided
protect the wound with a plaster
wear a lab coat, and put on protective gloves to handle any blood other than your own
wash your hands immediately if they accidentally come into contact with blood
never under any circumstance use a mouth pipette
spills of blood must be decontaminated immediately (ask a demonstrator)
at the end of the experiment dispose of glassware and anything which has come into contact with blood in
the sharps bin
Estimating Red Cell count
Dilute the blood 200x and place a layer 0.1mm thick on a special ruled haemocytometer slide (To dilute the
blood, 5 microlitres is added to 995 microlitres of HAYEM’S solution using a capillary pipette)
By counting those in a known area, the number or red cells in 1 litre of blood can be estimated
The white cells may also be counted, but as there are normally 500 RBC for each WBC, the consequent error
is negligible
Normal Range (Mean) RBC count:
o Male: 4.3 - 5.9 (5.1) x1012/l female: 3.7 - 5.3 (4.5) x1012/l
Measuring Haematocrit
Haematocrit (Hct) is the percentage of the volume of a sample of blood occupied by the red cells
This can be measured using a centrifuge
Normal Range (Mean) Hct %:
o Male: 40 - 52 (46) Female: 35 - 47 (41)
Measuing Hb concentration in whole blood
Method based on releasing the haemoglobin from the red cells and converting it to the stable derivative
CYANMETHAEMOGLOBIN
o Using modified Drabkin’s Cyanide-ferricanide solution
The absorbance of the diluted pigment is then measured spectrophotometrically
MCD Practicals Alexandra Burke-Smith
7
By examining in the same way a solution containing a standard quantity of the pigment (Hb), the absolute
concentration of the Hb in the blood sample can be deduced
Unknown Hb conc = (A of test solution x conc of standard) / A of standard solution
Normal Range (Mean) (g/dl):
o Male 13.3 – 17.7 (15.5) Female 11.7 – 15.7 (13.7)
Calculating the derived RBC parameters
MCV – mean cell volume
o = Hct / RBC count x 100
o Unit: fl (10-15 l)
o Normal Range (Mean): 80 – 100 (90) fl
MCH – mean cellular haemoglobin
o = (Hb x 10) / RBC count
o Unit: pg (10-12 g)
o Normal Range (Mean): 26 – 34 (30) pg
MCHC – mean cell haemoglobin concentration
o = (Hb x 100) / Hct
o Unit: g/dl
o Normal Range (Mean): 32 – 36 (34) g/dl
MDL2-2 – Mitosis Dr Peter Clark ([email protected])
1. Explain the process of mitosis and how it accurately distributes the replicated chromosomes between daughter
cells at cell division.
2. Identify and name in a microscopic specimen the stages of mitosis.
3. Explain how growing cells can be specifically targeted in chemotherapy.
Growing cells in culture
Much of our understanding of how cells grow and behave, as well as what goes wrong in cancer cells, has
come from these sorts of experiments.
The cells used in this practical derive from the frog Xenopus - they were chosen mainly because cells from a
cold-blooded animal will continue to grow and divide actively at room temperature in the practical
laboratory whereas mammalian cells would need to be maintained at 37C in special incubators. They are
also free from the potential health hazards associated with human cell lines which can carry viruses
infectious to people.
In this practical the cells are growing on the surface of coverslips placed in a Petri dish. The coverslips can be
easily removed, stained and mounted on slides for microscopic examination. The culture medium is a
complicated mixture of salts, nutrients, vitamins and other factors which promote growth and cell division.
o The pink colour is due to a pH indicator – it will go orange then clear if the medium is too acid, or
puce then purple if too alkaline.
Experiment Procedure
Experiment involves comparing uninhibited cells (control) with cells which have spent an hour in culture
medium which is identical except for the addition of an inhibitor
Inhibitor of cell division: VINBLASTINE
MCD Practicals Alexandra Burke-Smith
8
o Used as part of chemotherapy treatment of certain cancers; dose carefully regulated so that is
affects the most rapidly dividing cells, i.e. tumour cells
Staining:
o Briefly wash in phosphate-buffered saline (PBS)
o Fix in acid-alcohol (15 secs)
o Rinse in distilled water
o Stain chromosomes: one drop of neutral red stain
o Remove excess stain with tissue
o Examine under microscope
Identification of Stages of Mitosis
Interphase G2:
o Chromosomes not visible
o Nucleoli still present- DNA already replicated
o Nuclei stain weakly, except for the nucleoli
Prophase:
o Individual chromosomes within the nucleus begin to condense and take up stain.
o DNA replication has already occurred so each chromosome consists of two chromatids attached at
the centromere
o The nuclear envelope breaks down.
Metaphase
o Chromosomes maximally condensed (might be able to visualise X-shaped appearance)
o End of metaphase – chromosomes align on central plane of mitotic spindle; metaphase plate
Anaphase
o Sister chromatids of each chromosome separate
o Microtubules of the mitotic spindle attach to kinetochores on each side of the centromere, and pull
one chromatid to each pole of the spindle
Telophase
o Two separated groups of chromosomes begin to form the nuclei of the two daughter cells
o Chromosomes de-condense
o Nuclear envelopes re-form
o End of telophase – cytokinesis occurs when the rest of the cell divides (may be evidence of cleavage
furrow)
Interphase G1:
o Condensation process completely reversed
MCD Practicals Alexandra Burke-Smith
9
MDL1-3 – Enzyme kinetics
1. Draw the absorption spectra of NAD+ and NADH, and explain their use in enzyme assays.
2. Demonstrate how experimental values for reaction velocity at different substrate concentrations can be used to
construct a double-reciprocal plot (Lineweaver-Burk plot) of 1/V (velocity) against 1/S (substrate concentration) in
order to evaluate KM and Vmax.
3. Explain the effects of competitive and non-competitive inhibitors on KM and Vmax
Introduction
In this practical you will investigate a dehydrogenase enzyme, malate dehydrogenase. Dehydrogenases
oxidise their substrate with a parallel reduction of the cofactor NAD+ to NADH. (they can alternatively
catalyse the reverse reaction).
Spectrophotometry allows you to follow the enzyme reaction by the appearance of NADH (which absorbs at
340 nm) and hence analyse the enzyme kinetics.
You will measure the reaction rates at different concentrations of the substrate L-malate to determine the vmax and
KM for the enzyme. Malate dehydrogenase (MDH) catalyses the following reaction:
L-malate + NAD+ oxaloacetate + NADH
When NAD+ is in excess (i.e. when there is negligible product to undergo a back reaction) the initial rates of this
reaction conform to Michaelis-Menten kinetics with respect to the concentration of L-malate, and can be analysed
with a Lineweaver-Burk plot.
The rate can be measured easily in a spectrophotometer since the product NADH absorbs light at 340 nm whereas
the substrate NAD+ does not. The absorption obeys the Beer-Lambert law with a value of the extinction coefficient E
of 6.22 μmol-1.ml.cm-1 (see first practical for law)
Method
You are provided with: Reaction buffer, pH 9.5
Stock solutions of L-malate (0.5, 1, 2, 3, 10mM) and D-malate (10mM)
Stock solution of NAD+ (50mM)
Stock solution of MDH enzyme
Important: all of these except reaction buffer should be kept at ice temperature.
Carry out enzyme activity measurements as follows: 1. Ensure the spectrophotometer wavelength is set to 340 nm
2. Pipette the following into a 3 ml cuvette:
a. 1.75 ml reaction buffer
b. 0.15 ml NAD+ stock
1 ml of the relevant malate solution (start at the lowest concentration and work up)
3. Mix the contents by inverting twice, place the cuvette in the spectrophotometer and set the blank.
4. Now add 0.1 ml enzyme solution, mix as above and return to the spectrophotometer. Record the
absorbance immediately (zero time) and at 30 second intervals up to 3 minutes.
Work out the initial enzyme rate v by plotting absorbance A against time and converting to mol.min-1.ml-1 using the
extinction coefficient (the path length for these spectrophotometer cuvettes is 1 cm). You will also need to calculate
MCD Practicals Alexandra Burke-Smith
10
the concentration of malate [S] in the cuvette taking into account the volumes of other components. Finally draw a
Lineweaver-Burk plot of 1/v against 1/[S] to determine vmax and Km.
An example using real data is given below:
The rate obtained is given in absorbance change per minute, and this needs to be converted to
concentration units per minute.
The conversion factor is the
extinction constant E for NADH
which is 6.22 μmol-1.ml.cm
Beer-Lambert law A = E c ℓ
where ℓ = 1 cm (the length of the
light path through the cuvette),
you can simply rearrange the
equation to give: c = A/E. As an
example, the group above found
that with 2.0mM malate stock
(0.67mM final) the enzyme rate
was 0.250 absorbance units per minute. This means a rate of
change in NADH concentration (v) of 0.250/6.22 = 0.0402 μmol.ml-
1.min-1 and a value of 1/v of 25 μmol-1.ml.min.
Now the double reciprocal plot can be made: The kinetic parameters are obtained from the intercepts on the
axes.
o The intercept on the 1/v axis is 1/vmax
o The intercept on the 1/[S] axis is –1/KM
MCD Practicals Alexandra Burke-Smith
11
In this experiment there can be quite a lot of error in determining the intercepts, and taking the reciprocal
has the effect of magnifying errors in the measured rate, especially at the lower values of [S].
MDL1-4 – Analysis of data from Mitosis and Blood Cell practicals
1. Use simple statistical methods on real data sets generated in previous practicals to summarise the results for
reporting, as would be done in a scientific paper, and comment on the reliability of the results.
2. Understand how to quantify variation within a set of data, and to appreciate the factors that may lead to such
variation.
3. Use simple tests of statistical significance to compare two sets of data, e.g. with and without drug treatment
Calculations
1. Mode 2 (STATS)
2. Press option 1 (1-Var)
3. Enter data, pressing = after each number
4. Press AC
5. Press shift, then 1
6. Select option 5 (VAR)
Then…
7. for Standard deviation – select option 4 and press =
for the mean – select option 2 and press =
NB: (if you have calculated the standard deviation first, there is no need to re-enter all the data, just repeat from
step 4, or vice versa)
Standard Deviation
Measures the spread of the data about the mean
If the mean from two groups of data are plotted, with the corresponding standard errors, and there is NO
overlap – they are significantly different
MDL2-4 – Organisation of epithelial tissues
1. Identify individual cells at the light microscope level on standard histological sections.
2. Demonstrate the following on suitable light microscope histological sections: nucleus; nucleolus; cytoplasm;
position of cell membrane; cell in mitosis.
3. Classify epithelial types in an unknown specimen and be able to relate these to their probable function in the
body.
4. Identify the histological structures in terms of layers for the following tissues and explain how the epithelial
organisation defines function: oesophagus; small intestine; large intestine; trachea (but not rest of lung).
5. Recognise on a suitable transmission electron micrograph of a haematopoietic cell that it is non-epithelial.
6. Recognise the disruption of the normal cellular pattern in a familiar tissue of unambiguous examples of cancer.
Simple epithelium - cells are lined up in a single row, with their nuclei alongside each other
Basal lamina/basement membrane – the fibrous layer to which the base of each cell is attached (only just
detectable in the light microscope)
MCD Practicals Alexandra Burke-Smith
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Columnar epithelium – pink staining cytoplasm, function: absorption of the products of food digestion. Also
known as intestinal absorptive cells/enterocytes
Goblet cells – mucus secreting cells interspersed between epithelium with very pale cytoplasm and bulbous
shape
Brush border – close packed row of small finger-like projections of the apical membrane (ONLY) which serve
to increase the surface area; known as microvilli – required for cell polarisation
Junctions – between adjacent cells
Intestinal Epithelium
Epithelia can be described concisely in terms of their cell shape, arrangement and specialisation, using the
terminology above. Thus the epithelium lining the intestine would be described as a “simple columnar
absorptive epithelium with goblet cells”.
Lining epithelium separating the lumen and the rest of the body. If the lumen is small, the epithelium forms
a gland or duct.
Glands – tubular structure with secretory role. In the intestine known as crypts (simple tube with closed
end). Closed end: presence of secretory cells of the small intestine – the Paneth cells, recognised by the
brightly pink stained granules positioned assymetrically at the apical surface
NB: Types of gland Simple tubular – e.g. crypts; single closed tube
Compound – branched
Acina/alveolar – secretory cells restricted to regions with a specialised rounded shape at the ends of the
tubus
Mesentery
The mesentery is a thin layer of tissue that attaches parts of the intestines to the rest of the body. It allows
the whole length of the intestines to be folded up to fit within the abdominal cavity but still have a
connection carrying its blood and nerve supply, and it means that the intestines can be somewhat mobile
during peristalsis.
The epithelium is only one cell deep, in other words it is simple. Although you will not be able to see the cell
boundaries, you can guess roughly where they are since they will lie between nuclei. The cells are flat and
wide, something that is also reflected in the shape of the nuclei. Cells which are wider than they are tall are
known as squamous, and the epithelium surrounding the mesentery is described as a simple squamous
epithelium.
Another example of a simple squamous epithelium can be found on this slide. The epithelium lining blood
vessels (alternatively known as endothelium) is of the same pattern.
Oesophagus
The oesophagus has an epithelium lining the central lumen which in this case is stratified, meaning that it is
many cells thick.
The cells stain differently according to their level with those nearest the basal lamina being almost purple,
those higher up having a deep pink cytoplasm, and those nearest the lumen having a very pale cytoplasm.
The individual cells have a range of different shapes with basal cells being almost rounded but those closer
to the lumen (the majority) being squamous. The structure is referred to as a stratified squamous
epithelium.
The arrangement is designed to line the lumen of the oesophagus in a way which is resistant to damage. The
stratified arrangement means that if the cells adjacent to the lumen become damaged, the cells underneath
maintain the function of the epithelium as a barrier and can divide to replace those that are lost.
MCD Practicals Alexandra Burke-Smith
13
The body uses a stratified squamous epithelium in several locations as a protective strategy, with the skin
being a special case. Skin has to be waterproof to prevent the cells below drying out and dying. The surface
cells of the epithelium have died (“cornified”) leaving only the dense network of their keratin-containing
intermediate filaments to form a non-cellular protective layer that absorbs oily secretions to become
waterproof.
Trachea
The epithelium separates the lumen from the surrounding body tissue, including rings of cartilage whuch
hold the airway open
You should be able to find a region where the plane of section is approximately right angles to the basal
lamina and you can identify individual columnar cells. On their apical surface are fibrous projections that are
much larger than the brush border of the intestine: these are cilia. Interspersed among the ciliated cells are
goblet cells (with this stain they are purplish). Goblet cells produce mucus which lines the airway and
protects it from drying out. The cilia beat to move the mucus up towards the back of the mouth so that the
mucus doesn’t accumulate.
Although you can identify individual columnar cells you will find that their nuclei are not lined up neatly as
they were in the small intestine. Some cells don’t stretch all the way to the luminal surface of the epithelium.
This type of epithelium is referred to as pseudo-stratified because the nuclei look as if they are arranged in
layers. It is not actually stratified because all of the epithelial cells have their base attached at the basal
lamina. Overall this epithelium is referred to as a “pseudo-stratified columnar ciliated epithelium”.
NB: Other types of epithelium Cuboidal – cellsof approx. equal height and width, which are often present in ducts but are functionally
unimportant
Urinary – certain features of both stratified and pseudo-stratifies (sometimes called transitional);
speacialised to be highly impermeable to urine and to be able to stretch as the bladder fills
Cell renewal in epithelia
The cells in a pseudo-stratified epithelium which do not extend all the way to the luminal surface a specialised
population of dividing cells, known as stem cells. Other cells are formed by division of stem cells, and do not
themselves divide. In the small intestine stem cells are located within the crypts and in this case their daughter cells
can form enterocytes which become specialised for absorption and move out of the crypt to cover the villi. The same
strategy for cell renewal is found in stratified epithelia where the dividing stem cells are present in the basal layer
adjacent to the basal lamina. In all three cases the dividing cells are located where they have minimum susceptibility
to damage.
A different strategy is found in some other epithelia, including simple squamous and the simple cuboidal epithelia of
many ducts. Here all of the cells are capable of cell division when required.
MCD Practicals Alexandra Burke-Smith
14
MDL1-5 – Microbiology practical
1. This practical aims to introduce the microbiology diagnostic tests which are commonly used in a hospital
laboratory to identify medically important pathogens. Having completed the practical you will understand, and be
able to use:
2. The Gram stain to distinguish different types of bacteria
3. Recognise the Gram stain characteristics of important pathogens including Staphylococci, Streptococci, Neisseria
and Enterobacteriaceae such as Escherichia coli
4. A microscope to examine slides of microorganisms
5. Selective media and common biochemical tests to identify pathogens
6. Microbiology diagnostic laboratory flowcharts to identify pathogens
7. Antibiotic sensitivity testing and choose suitable antibiotics to treat an infection
Bacterial pathogens can be partially identified by their staining reaction, shape and arrangement (e.g. single cells,
clusters, chains). Most bacteria can be stained using Gram’s Method and classified as Gram-positive or Gram-
negative. Microscopes have been set up in this MDL section to view fixed bacterial preparations stained by Gram’s
Method.
Gram positive bacteria tend to stain BLUE. Gram negative bacteria tend to stain RED. Most bacterial infections are diagnosed by growing the causative organism on an appropriate selection of
bacteriological media. Typically these are agar based and may contain, for example, whole or lysed blood as a
nutrient, or substances such as bile salts as an inhibitor or phenol red as an pH indicator. You are provided with a
selection of agar plates on which have been seeded various species of bacteria.
MDL2-5 – Connective tissues and muscle
1. Explain the range of connective tissue types in terms of the different types of extracellular matrix components
secreted by different cell types.
2. Explain the way in which the strength of the skeleton is maintained throughout life in terms of the cellular
processes of bone deposition and mineralisation, and bone resorption.
3. Give examples to illustrate the different means by which cells receive nutrients and O2 in different connective
tissues.
4. Describe the following tissue types, giving for each an example of its location in the body: adipose tissue, loose CT,
dense irregular CT, dense regular CT, cartilage, bone, striated muscle, smooth muscle.
5. Recognise the characteristic microscopic appearance of the above tissues when they are present in association
with epithelial structures in histological slides of the various organs of the body.
6. Recognise the disruption of the normal cellular pattern in a familiar tissue of unambiguous examples of acute
inflammation.
Connective tissue
The general name given to a family of tissues including cartilage, bone, tendons and ligaments as well as those commonly referred to as loose or dense connective tissue. Common features include:
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The support cells which maintain the tissue are not attached to one another (i.e. unlike epithelial cells).
The majority of the volume in most (though not all) connective tissues is taken up by extracellular matrix.
Other cells may be present, for example those with immunological functions.
The tissues are derived from mesenchyme, an embryonic tissue principally associated with the mesoderm
layer of the early three layer embryo.
However the support cells are specialised to the particular tissue, and in particular to producing extracellular matrix (ECM) with the necessary properties. In all cases the ECM components can be reduced to three types:
Fibrillar structures which provide strength against tensile forces applied to the tissue; main protein
components are collagen, elastin and fibrillin.
Viscous liquid component which provides resistance to compressive forces. The main molecular components
are complex negatively charged polysaccharides which bind water and Na+ ions and are mainly
glycosaminoglycans or proteoglycans. The liquid component also provides a solvent in which oxygen,
nutrients, waste products and small signalling molecules can diffuse.
Other molecules whose main function is in binding to cells via specific cell surface receptors and mediating
their attachment to the ECM. The best known example is fibronectin.
Dense and loose connective tissue
The outside surface of the skin can be
recognised because it is covered with an
epithelium (called the epidermis) whose cells
stain a pale blue colour.
Below this is a layer of dense connective
tissue called the dermis. Individual cells are
separated by pink staining ECM.
The fibrous components are more clearly seen
in the loose connective tissue found
deeper down in the skin. The fibres are less
closely packed and you will see that both thick
and thin fibres are present.
Look at the cellular components of the loose connective tissue. The support cells for both dense and loose
connective tissue are known as fibroblasts, and they have a spindly shape with correspondingly pointed
looking nuclei. Other cells are mainly migratory white blood cells such as lymphocytes (which are
especially abundant where the tissue is likely to be exposed to foreign substances, e.g. the loose connective
tissue underlying the intestinal epithelium) and neutrophils (which become abundant during an acute
inflammatory response). Two types of cell which are normally resident in loose connective tissue but are
related to white blood cells are the macrophages (corresponding to blood monocytes) and mast cells
(corresponding to basophils).
Dense connective tissue also has fibroblasts as its support cell type, responsible for synthesising the ECM.
The dense connective tissue of skin is known as dense irregular connective tissue because the fibres
are randomly oriented to give the tissue equal strength in all directions. In some locations – such as tendons
and ligaments – strength is required in a single direction, and in this case the collagen and other fibres are
aligned in parallel and the tissue is known as dense regular connective tissue.
Part of the loose connective tissue in the skin contains a large number of fat cells or adipocytes. These
appear white and large – about 10 times the diameter of most other cells. In fact the majority of the cell is
taken up by a single large vacuole containing lipid with all the rest of the cell (nucleus, cytoplasm, organelles)
squeezed in a thin rim around the outside. The ECM components of the tissue lie between the fat cells.
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Cartilage
Appear as large purple staining regions easily identified at low magnification.
At higher magnification look for the support cells, recognising them by their blue staining nuclei. The support
cells of cartilage are called chondrocytes, and they are well separated from each other. They each lie in
their individual pale staining regions, called lacunae. The remaining space, staining purple, is the ECM of
the cartilage. You will find it difficult to see evidence of the structure of the ECM since the dense fine
network of collagen fibres and the glycosaminoglycans rich in sulphated sugars produce a fairly homogenous
appearance in this type of cartilage which is called hyaline cartilage. Other types of cartilage have
modified ECMs. Fibrocartilage contains thicker collagen fibres oriented to provide tensile strength in a
particular direction, for example in the pubic symphysis (which is the joint connecting the two halves of the
pelvis at the front) or the intervertebral discs (connecting adjacent vertebrae of the spinal column. Elastic
cartilage contains additional elastic fibres to provide pliability and resilience (for example in the external
ear).
Cartilage is unusual in that it has no blood vessels and substances reach the chondrocytes by simple diffusion
through the ECM – despite the apparent solidity of cartilage it consists of about 70% water.
Bone
Bone is a specialised connective tissue where the ECM (termed osteoid) has become mineralised with
calcium phosphate crystals to provide great compressive strength.
The relatively solid region around the edge of the bone shaft is known as compact bone or dense bone.
Within it there are the support cells of bone, called osteocytes, within lacunae. There are also other
cavities containing blood vessels. The mineral prevents free diffusion, and small channels called canaliculi
provide a pathway for diffusion of O2 and nutrients from the blood vessels to the osteocytes. Slide 14211 has
been specially stained to show up osteocytes and canaliculi. In transverse section on that slide it can be seen
that bone is arranged in structural units called osteons or Haversian systems which have a central canal
containing blood vessels, with the ECM of the bone arranged in concentric layers around this central canal.
The collagen fibres are oriented differently in each layer to provide additional strength.
The central part of a bone has spaces between the
bone tissue, and is known as spongy bone or
cancellous bone. The bony structures here are
called trabeculae, and they are usually arranged
in the way that best resists the mechanical stresses
on the bone. Most of the space between the
trabeculae is filled with bone marrow, the site of
developing blood cells. There may also be fat cells.
New bone is laid down by cells called osteoblasts
and bone is reabsorbed by cells called
osteoclasts (these cell types are not easily
identified in histological sections). Normally the two processes are in balance but they allow deposition of
new bone to be adjusted to strengthen bone in regions of mechanical stress or to heal a wound. There can
be net bone production during growth, or net resorption resulting from disuse in immobile patients or from
osteoporosis. Because the mineral component of bone can act as a store for Ca2+ and phosphate, these
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activities are under hormonal control from calcitonin and parathyroid hormone to maintain the normal
blood concentrations of these ions.
Most (though not all) bones start as cartilage precursors in the foetus, which then get converted to bone – a
process known as endochondrial ossification. In long bones, a cartilaginous region persists at the
growth plates between the shaft and heads until the bone has reached its adult length. Cartilage also lines
the bone surface where it forms part of a joint. Elsewhere the bone is lined with a layer of dense connective
tissue – the periosteum.
Muscle
The majority of muscle in the body is
skeletal muscle, connected to part of the
skeleton and acting across a joint to move
one bone in relation to another. This is an example of a striated muscle, named for the striped
appearance due to the repeating sarcomeres that make up the contractile apparatus.
By comparing the transverse and longitudinal sections work out how the muscle is made up of a bundle of
myofibrils, each containing multiple nuclei arranged around their perimeter. These are made up of a large
number of precursor cells which have fused together to make a syncytium in which the contractile
apparatus in the cytoplasm becomes continuous. Attached round the edge of each myofibril is an ECM
containing collagen fibres that are ultimately continuous with those of the tendons or bones on which the
muscle acts.
Cardiac muscle (which you will study in more detail next term) is also a form of striated muscle, but in this
case the muscle cells remain separate and are connected end to end at structures called intercalated
discs. Some cardiac muscle cells have become specialised to form the conducting system of the heart. (In
contrast skeletal muscle is controlled by innervation from motor neurons).
Smooth muscle is the form associated with organs and vessels, and it is controlled by the autonomic
nervous system. The wall of the bladder is made up from individual cells which can be hundreds of
micrometers in length, and which have a central nucleus with a characteristic cigar shape, appearing circular
in transverse section. The contractile apparatus is differently organised from striated muscle, with no
repeating units being visible. Smooth muscle is not capable of the rapid powerful contraction of skeletal
muscle, but can contract to a smaller fraction of its relaxed length and can maintain contraction for very
much longer times.